1. Field of the Invention
The present invention relates to high throughput assay systems and methods for identifying agents that alter the level of expression of cellular proteins, with special emphasis on integral membrane proteins in mammalian cells.
2. Background of the Related Art
The ability to respond to the surrounding environment and the control of entry and exit of molecules through the cell membrane are fundamentally important functions of any mammalian cell. These functions are due, in significant part, to the various proteins that reside, in whole or in part, within the cell membrane.
The cell membrane of mammalian cells is relatively impermeable to water-soluble substances, such as ions, small inorganic molecules, peptides and proteins. To enter and/or influence a cell, such hydrophilic substances must interact with at least one protein (e.g. a receptor, an ion channel or a transporter) that resides in the cell membrane and is exposed, at least in part, on the cell surface to the extracellular milieu. In contrast, lipophilic substances, such as steroids, can diffuse directly through the cell membrane into the cytoplasm where it can then interact with one or more target proteins.
There are two basic responses from a cell when an external stimulatory molecule (e.g. a peptide or an organic or inorganic molecule) interacts with a cell surface protein: (i) an ionic or molecular (or macromolecular) material is vectorally transmitted from the outside of the cell membrane to the cytoplasm inside the cell by transport through the lipid bilayer and vice versa; and/or (ii) a signal is transmitted to the cytoplasm or a protein therein by virtue of a change in the membrane protein, e.g. a change in the conformation of the membrane protein and/or the state of aggregation of the membrane protein.
The transfer of a signal across the cell (signal transduction) begins with the binding of an extracellular substance (ion, small molecule, protein) to an extracellular domain of a protein resident in the cell membrane. Binding of the substance to an extracellular domain of the transmembrane protein causes the protein to change form an inactive to an active form. This active form then stimulates catalytic activity, or some similar such response, that generates a cytosolic signal (which is sometimes in the form of one or more secondary messenger substances in the cytoplasm).
There are two major types of such signal transduction in mammalian cells: (i) the transmembrane protein may have a protein kinase activity in its cytosolic domain, the activity of which is activated when the extracellular substance binds to the transmembrane protein (the kinase then phosphorylates its own cytoplasmic domain, which enables the transmembrane protein to associate and activate another protein, which in turn acts on other proteins and substances within the cell cytoplasm); and (ii) the transmembrane protein may interact with a G protein that is associated with the membrane, which causes the GDP (guanine diphosphate) bound to the G protein to be replaced by GTP, resulting in dissociation of the G protein into monomer and dimmer fragments, one or both of which, in turn, acts upon a target protein (also often associated with the membrane, requiring it too then to act upon yet another target protein, this one in the cytoplasm).
The physical transfer of material across the cell membrane permits a wide range of substances to get into and/or out of a cell, including ions, small molecules (such as sugars and hormones) and macromolecules (such as proteins and enzymes). Three major routes exist for such material transfer(s): (i) proteins resident in the cell membrane may form channels that permit the passage of ions, such as sodium, potassium and chlorine, from the extracellular milieu through the membrane and into the cytoplasm, or vice versa; (ii) proteins resident in the cell membrane may bind small molecules, such as sugars, on one side of the membrane and then release that same molecule on the opposite side of the membrane, thereby acting as transporters; and (iii) proteins resident in the cell membrane may bind small molecules and so trigger the process of internalization, in which the bound protein:molecule pair is brought into the cell by endocytosis (at some point, the protein:molecule pair becomes separated; the protein may then be returned to the cell surface to interact with another small molecule or it may be degraded).
Common features of all these proteins include their relatively, large size, the multiple hydrophobic regions spanning the cell membrane, and hydrophilic extracellular and/or intracellular domains.
With respect to the passage or trafficking of macromolecular substances, proteins begin the pathway that leads to secretion by co-translational transfer directly from ribosomes to the membranes of the endoplasmic reticulum. These proteins are then transferred to the Golgi apparatus, where they are sorted according to their final intended destination and move towards the cell membrane.
More specifically, proteins enter the ER during synthesis and are folded and glycosylated, at least partially. The proteins are then transferred to the cis face of the Golgi apparatus (proteins that are to be resident in the endoplasmic reticuluim are returned to the ER at this time). Further glycosylation occurs as the proteins move through the Golgi stacks from cis to trans. Specific signals cause some proteins to be returned to the ER, some proteins to be retained in the Golgi, some proteins to be transported to endosomes and lysosomes, and some proteins (cell surface proteins) to be transported to and retained in the cell membrane.
Those proteins that are transported to and retained in the cell membrane follow the longest and most extensive trafficking pathway, entering the membranes of the endoplasmic reticulum and subsequently traveling through the membranes of transition vesicles, the Golgi complex and secretory vesicles before reaching their final destination. Among these proteins are active and passive transport proteins and cell surface receptors.
To illustrate the complexity of the trafficking pathway, in epithelial cells lining body cavities, the sorting and distribution mechanism of the ER places different proteins in distinct subparts of the cell membrane. For example, proteins transporting sugars and amino acids in intestinal epithelial cells are distributed to that region of the cell membrane facing the intestinal cavity. MHC molecules and the poly-Ig receptors binding antibodies wind up in segments of the cell membrane of epithelial cells on the side opposite the side facing the body interior.
The following is a table of some known cell surface molecules, receptors and membrane-associated proteins.
EGFRepidermal growth factor receptorβ-adrenoreceptorGPCRGABAaRGABAa-gated ion channelnAChRACh-gated ion channelP-glycoproteinmembrane channelKir2.2ion channelMCRmelanocortin receptorhERGion channelA4TM4 familyAbc2ABC transporter; multi-tm; mdr subfamilyAcPLrequired for IL-18 receptor signaling. Contains two IgdomainsAF1qfused to MLL in some leukemiasAlpha-6 integrincomplex with NAG-2Alpha-9 integrinMediates cell-cell and cell-extracellularmatrix interactionsART-4Adenocarcinoma antigen recognized by T-lymphocytesB29Ig superfamilyBAP31potential membrane proteinBeta ig-h3TGF-beta induced; may be associatedwith microfibrils and the cell surfaceBgpdMay play role in self-renewal/diff. of epitheliaCatechol-O-membrane-bound form; inactivates catecholaminemethyltransferaseneurotransmittersCD9TM4; with CD19 in multimolecular B1-integrincomplex, also NAG-2CD19Ig domains; B-cell growth regulationCD27receptor for CD70; TNFR-family; apoptosisCD31/PECAM-1adhesion moleculeCD34“Stem cell antigen”CD37TM4 on B-cellsCD48ligand for CD2CD53TM4 superfamily; high in Burkitt lymphoma cell linesCD54/ICAM-1adhesion moleculeCD59complement inhibitory proteinCD69Involved in lymphocyte proliferationCD87/PAR2Urokinase plasminogen activator receptorCG1Possible TM4 cell surface proteinCoronin-2WD40 domainDPH2Lsingle tm; ovarian ca. suppressorDR5Death receptor for TRAIL; apoptosis-inducingEBI17-tm receptorEBI3contains FnIII domainEP2 or -4prostanoid receptorEvi2BImplicated in leukemogenesisFC gamma R IAntibody Fc receptorFZD4Frizzled 4; receptor for Wnt family ligandsFlk-2receptor tyrosine kinase; differentially expressedFlotillinBAND7 familyGITRglucocorticoid-induced; TNFR family; apoptosis assocGluR3Glutamate ReceptorGlypicanMajor heparan sulfate proteoglycanGPCROrphan G-protein coupled receptorGPR-9-6/CCR9orphan 7-tm hormone receptorHEM-1TM4 familyHepatocytemembrane-bound formGF activatorinhibitorIB3089APutative transmembrane proteinICAM-2Intercellular adhesion moleculeIL-2-gammacommon chain for IL2 and 4 and 7 and 13IL-3-betacommon to IL3 and 5 RIL-3RHematopoietic growth factor receptor associatedwith survival and differentiationIL-4-alphamature form includes IL2 gamma chainIL-6RHematopoietic growth factor receptorIL-7RB-cell growth factorJTBCell surface protein; rearrangedin a jumping translocationL-selectinlymphocyte homing moleculeLAPTm5may have functional role in embryogenesisLigatintrafficking receptor for phosphoglycoproteinsLOX-1Lectin-like oxidized low-density lipoprotein receptorLSM-1interacts with CD45 on lymphocytesLymphotoxin-b Rposs. function in immune developmentMac-2MamaScavenger-like Cys-rich domainMb-1supposedly B-cell restricted; CD3-likeMcp-1chemokine RMDC15metalloprotease-disintegrin; tm glycoproteinMEGF9EGF repeatsMIP-1aRchemokine RMitsugumin 23TM4 protein on ER and nuclear membranesMP709 tmNAG-2Surface TM4 protein similar to CD53;forms complexes w/integrinsNET-4TM4 proteinNET-6TM4 proteinNeuropilinSemaphorin III receptor; also binds VEGFNHE-1sodium/hydrogen antiporterNotch-1Required for the correct differentiation of many tissuesPAR-1 or -2Plasminogen activator receptorperlecanbasement membrane heparan sulfate proteoglycanPft27Putative 7-tm receptorPIRA-1Ig-like; suggested immune regulatory roleProstaglandin E R7-tm receptorProtocadherin-2CExpressed in developing brainRPTP-sigmareceptor tyrosine phosphatase; contains Fn III domainsSelenoprotein RPutative; contains domain of unknown functionSemaphorin BGrowth cone guidance proteinSIMstromal cell protein; TM4 surface RSmoothenedRecptor for Sonic Hedgehog; 7-tmSortilinneurotensin R (NT converting enzyme is in stroma)Stromal CellPotential TM4 cell surface proteinProteinSYBL1synaptobrevin-likeTLR2Toll-like receptor 2TLR4Toll-like receptor 4TSA-1 (Sca-2)Thymic shared antigen; also called Ly-6ETspan6TM4 superfamily; unknown function
The identification of compounds that alter the level of expression of one or more of these proteins is therefore quite important from a number of different perspectives and with a number of goals in mind. For example, identifying a compound that alters the level of expression of a particular protein may lead to the discovery of new therapeutic agents. Alternatively, such an identification may lead to the discovery of the cause or pathway of a defect or disorder. In that way, entirely new areas of research are opened up, as well as a wealth of previously-unknown targets for potential therapeutic intervention.
Moreover, the identification of compounds that alter the level of expression of one or more of these proteins should include compounds that directly or indirectly affect the level of protein expression. A compound that alters the level of expression of an integral membrane protein may do so directly, for example, by binding directly to the protein and inhibiting trafficking. Alternatively, a compound may alter the level indirectly, for example by acting on one or the chaperones that facilitate transport and integration of membrane proteins in the cell membrane or by acting on a protein that degrades the target membrane protein.
There therefore exists a need in the art for assays that can identify compounds that alter the level of expression of proteins, particularly integral membrane proteins, as well as the mechanism by which that level is altered.